roleofthe gut pathophysiology

Gut 1996; 39: 587-593

Role of the gut in the pathophysiology ofextrahepatic biliary obstruction

W D B Clements, R Parks, P Erwin, M I Halliday, J Barr, B J Rowlands

AbstractBackground-Gram negative septic eventsare the commonest source of morbidityand mortality as a result of surgery injaundiced patients. The large intestineprovides the major source of Gramnegative bacteria in mammals and isimplicated in the pathogenesis ofsystemicendotoxaemia in obstructive jaundice.Bile salts have an important part inmaintaining indigenous microecologicalhomeostasis through their emulsifyingproperties.Aims-The aim was to investigate theeffects of biliary obstruction and isolatedexternal biliary diversion on gastro-intestinal structure and caecal bacterialflora in relation to bacterial trans-location.Method-Six groups of adult male Wistarrats were studied (no operation, shamoperated, and bile duct ligated (BDL) forone and three weeks and a choledocho-vesical fistula (CDVF) for one week). Atthe end of the study period plasma wasassayed for evidence of endotoxaemia andthe animals were tested for bacterialtranslocation to the mesenteric lymphnode complex (MLNC), liver, lungs, andspleen. Quantitative and qualitativebacteriological studies were performed onthe caecal contents and segments of colonand terminal ileum were washed andprepared for histological assessment.Results-Bacterial translocation was sig-nificantly increased in the BDLl (68.8%)and BDL3 (60%) groups compared withthe shaml (6.3%), sham3 (9. 1%), Nooperation (0O/o), and CDVF1 (16.7%)groups. Although translocation was morepronounced in the BDL1 group, this wasalmost exclusively to the MLNC comparedwith the more widespread translocation toother organs in the BDL3 group. TheBDL3 group was the only group withsignificantly raised concentrations ofendotoxin and anticore glycolipid. Thecaecal Gram negative aerobic counts weresignificantly increased in the BDLl andCDVF1 groups compared with all othergroups. There was evidence of structuralabnormalities in the terminal ileum ofrats jaundiced for three weeks, but not inthe other groups.Conclusions-Biliary obstruction for oneand three weeks promotes bacterial trans-location although the mechanisms may bedifferent. Absence of intralumenal bileresults in a significant but self limiting

increase in the Gram negative aerobicpopulation, which may account for trans-location in the early stages of biliaryobstruction. As the duration of biliaryobstruction increases systemic endo-toxaemia is a consistent feature which,combined with factors such as immuno-logical depression and physical disruptionof gut barrier function, may promotebacterial translocation perpetuating sys-temic sepsis.(Gut 1996; 39: 587-593)

Keywords: bacterial translocation, jaundice, sepsis, bilesalts.

Perioperative Gram negative septic eventscomplicate the outcome of jaundiced patientsundergoing invasive diagnostic and therapeuticprocedures. 1-3 Systemic endotoxaemia is impli-cated in the high morbidity and mortality inthis patient population.4 5 The mechanism ofendotoxaemia and the pathophysiologicalsequence of events in obstructive jaundiceremains unclear.The two main physiological components of

extrahepatic biliary obstruction are firstly,obstruction of the biliary tree with a con-comitant rise in intraductal biliary pressure,and secondly, interruption of bile flow to thegastrointestinal tract. Mononuclear phagocyticfunction is depressed in obstructive jaundice6-8and some authors have shown the passage ofviable enteric bacteria to normally sterile extra-intestinal sites in murine models of biliaryobstruction.9-10 Both mechanisms are probablycomplementary in the development of systemicendotoxaemia. This study investigates theeffects of increasing duration of biliaryobstruction and external biliary diversion onthe indigenous microecology of the gastro-intestinal tract in relation to bacterialtranslocation.

Methods

Experimental designAdult Wistar rats (250-300 g) from ourbreeding colony were housed in groups of fourunder constant temperature (22°C) andhumidity with 12 hour dark/light cycles andallowed standard laboratory animal feed(Robert Morton and Co Ltd, Ballymena, UK)and water ad libitum at all times throughoutthe experimental period. Rats were assigned toone of six groups: control (no operation), bileduct ligation, (BDL) and sham operated for a

Departments ofSurgery andMicrobiology, theQueens University ofBelfast, Institute ofClinical Science,Grosvenor Road,Belfast BT12 6BJ, UKW D B ClementsR ParksP ErwinM I HallidayJ BarrB J RowlandsCorrespondence to:Mr W D B Clements,Department of Surgery, ICS,Grosvenor Road, BelfastBT12 6BJ, UK.Accepted for publication28 May 1996

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Clements, Parks, Erwin, Halliday, Barr, Rowlands

period of one or three weeks (control (n=14),BDL1 (n=16), shaml (n=16), BDL3 (n=10),sham3 (n=11)), and a further group wasstudied one week after sterile external biliarydiversion by means of a choledochovesicalfistula (CDVF1 (n=4)).

Operative proceduresAll procedures were performed observing strictasepsis under general anaesthesia, establishedusing intramuscular ketamine (6 mg/100 g;Parke-Davis Veterinary, Gwent, UK) andxylazine (0.7 mg/100 g (Bayer UK Ltd, BurySt Edmunds, UK). The method described byLee et al was employed for bile duct ligation.In brief, the common bile duct was mobilisedin the upper abdomen via a 1 cm incision,doubly ligated using 5-0 silk, and divided."Sham operated rats had the bile duct mobilisedbut not ligated. The choledochovesical fistulawas performed in a fashion originally describedby Diamond and Rowlands in which a finebore silastic cannula (internal diameter 0O51mm, external diameter 0.94 mm, Dow-Corning, Michigan, USA) was introduced intothe proximal bile duct and doubly ligated insitu with 5-0 silk sutures.'2 Once there wassatisfactory bile flow from the distal end of thecannula, the cannula was inserted into thedome of the bladder and sutured in place usinga 5-0 silk purse string suture. All abdominalincisions were closed in two layers using 3-0chromic catgut.At the end of the study period animals were

deeply anaesthetised, 2 ml ofvenous blood wascollected in glass endotoxin free tubes andplaced on ice before centrifugation (15 minutesat 2000 g) at 40C. Plasma samples werealiquoted and stored at -700C for subsequentassay. Animals were tested for bacterialtranslocation and small and large intestinesampled for histopathological examination.

Testingfor translocation of bacteriaUnder general anaesthesia a midline lapar-otomy was performed and swabs for bacterio-logical analysis were taken from the peritonealcavity. Subsequently 3 ml blood was collectedfrom the portal vein for aerobic and anaerobicculture using the radiometric Bactec 460(Becton Dickinson, Towson, MD, USA).Blood cultures were read on days 1, 2, 4, and7 at a threshold of 20 and 30 for anaerobes andaerobes respectively. Positive blood cultureswere plated out on appropriate media andidentified by standard bacteriological tech-niques.The right lobe of the liver, and the spleen,

right lung, mesenteric lymph node complex,and caecum were removed and each placed inpreweighed sterile glass bottles containingsterile prereduced brain heart infusion (BHI)medium. The bottles were transferred to ananaerobic cabinet (MK3 anaerobic workstation, Don Whitley Scientific Ltd, Shipley,Yorkshire, UK) in which tissue homogenateswere prepared in 2 ml BHI using sterilemortars and pestles and a homogeniser (Sorvall

Omnimixer, Ivan Sorvall Ltd, Norwalk, CT,USA). Serial dilutions of the caecal homo-genate ranging from 10-3 to 1O' were preparedin prereduced BHI. A total of 100 ,u aliquotsof homogenates were cultured on blood agar,chocolate agar, McConkey agar, tryptic soyagar, and in cooked meat broth. All platesexcept those for culture of the strict anaerobeswere removed from the anaerobic cabinet andincubated at 37°C for 48 hours. Anaerobeswere cultured for five days. Aliquots of brothwere plated out the next day on the fourstandard agar plates. After the appropriateincubation periods, individual colonies werecounted and each organism plated out forpurification. Organisms were identified andquantified as colony forming units /g tissue(cfu/g) using the formula:

NXDX2X 101Wwhere N=number of colonies on the plate;D=dilution inoculated on the plate; W=weightof specimen/g; 2=2 ml BHI; 10=innoculum(0 1 ml).

Histological assessmentA 1 cm segment of ascending colon andterminal ileum was excised, lavaged withnormal saline, and then fixed with 3%glutaraldehyde, processed by conventionalmethods, and embedded in paraffin wax.Ultrathin (1 ,um) sections were cut and stainedby haematoxylin and eosin and examined witha Leitz Laborlux K microscope.

Plasma assaysBilirubin concentrations were assayed using astandard biochemical technique and expressedin ,imol/1. Endotoxin concentrations wereassayed using the quantitative Limulus lysatechromogenic assay (Coatest endotoxin, KabiDiagnostica, Molndal, Sweden) and expressedin pg/l. The samples were pretreated by a1 0-fold dilution in pyrogen free water and heattreatment for five minutes at 85°C to negatethe effects of plasma inhibitory factors on theassay. Endotoxin present in the plasma con-verts a proenzyme to an active enzyme, whichacts on a chromogenic substrate producing acolormetric change, detectable spectrophoto-metrically at an absorbence wavelength of 405nm.

Anticore glycolipid antibody concentrations- the relative concentration of antibodies to thecore glycolipid region of lipopolysaccharide -were measured using an enzyme linkedimmunosorbent assay (EndoCab, Celltech,Slough, England). This technique, originallydescribed by Scott and Barclay, employsmicrotitre plates coated with a cocktail of fourrough endotoxin strains complexed withpolymyxin B sulphate. ' Prediluted sampleswere incubated with the solid phase, andbound rat IgG detected using a specific antiratIgG-peroxidase conjugate (Serotec Ltd,Oxford, England). The results are expressed asa percentage of control values obtained fromnormal rats.

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Role of the gut in the pathophysiology of extrahepatic biliary obstruction

TABLE I Summary of results displaying the incidence of bacterial translocation, caecal Gram negative aerobic counts,systemic concentrations of bilirubin, endotoxin, and anticore glycolopid antibody (ACGA)

Model No operation Sham 1 BDLl CDVFI Sham3 BDL3

BT (%) 0 6-3 68-8** 16-7 9.1 60*Bilirubin (umol/1) 1-5 (0.6) 1 1 (0 1) 151 (4-1)** 1-35 (0.3) 1-3 (0.1) 147 (7.4)**Endotoxin (pg/ml) 0.5 (1-2) 1.7 (1 1) 26-9 (20.0) 1-6 (1-6) 1-5 (0.8) 34-5 (16)**ACGA (% increase) 92 (35.7) 109 (11) 163 (33.8) 129 (8.7) 98-4 (10-3) 239 (37.5)**Gram negative aerobes 5-93 (0 22) 5.52 (0.24) 6-52 (0 30)* 7-17 (0.45)** 5-34 (0.35) 5-44 (0.50)(-log ,o cfu/g)

Results are mean (SEM). *p<0.05; **p<0 01.

Statistical analysisData analysis was performed on an OlivettiM300-30 microprocessor using Arcus pro-fessional software (Iain Buchan, Oxford, UK).Non-parametric statistical analysis (Fisher'sexact test, Kruskal-Wallis, and Mann-WhitneyU tests) was used throughout and significanceaccepted at the 5% level.

Results (Tables I, II, and III)

General changes and plasma assaysRats undergoing bile duct ligation wereclinically jaundiced within three to four daysand were maximally jaundiced by one weekwith conjugated bilirubin present in the urine.Animals jaundiced for one week were sprightlywith similar energy levels to controls and noovert evidence of ill health despite experiencinga more pronounced initial weight loss aftersurgery (Fig 1). With increasing duration ofbile duct ligation animals were less energeticalthough appetite and weight gain were similarto those of control rats (Fig 1). Rats jaundicedfor three weeks had significantly increasedserum concentrations of both endotoxin andanticore glycolipid compared with the shamoperated group at three weeks (p<0001,Mann-Whitney U test). Although there weresporadic increases in both endotoxin and

TABLE II Prevalence of translocating organisms seen in the various groups expressed as thenumber of episodes in which an individual organism was recovered and its percentage oftotal translocation episodes within individual groups

Translocating organism BDLl BDL3 Shaml Sham3 CDVFI

Ecoli 16 (73) 3 (20) 2 (100)Gram +ve bacillus 2 (9) 5 (33)Gram -ve bacillus 1 (4.5) 1 (6.7)Enterobacter species 1 (4.5) 1 (6-7) 1 (100)Streptococcus species 2 (9) 2 (13-3)Moraxella species 1 (6.7)Pasteurella species 1 (6.7) 1 (50)Acinetobacter species 1 (50)Anaerobic bacillus 1 (6.7)

Values in parentheses are /.

TABLE sM Qualitative alterations in the caecal Gram negative bacteria displayed aspercentage prevalence seen in control groups andjaundiced rats

Organism Control Shaml Sham3 BDLl BDL3 CDVFI

Ecoli 83 100 67 100 100 100Pseudomonas 17 22 17 63 33 83Pasteurella 50 11 33 75 33 67Shigella 0 11 0 25 0 33Moraxella species 17 0 67 13 50 33Kiebsiella species 17 0 0 0 33 17Enterobacter species 0 0 17 0 25 0Proteus species 0 0 0 0 8 0Acinitobacter 17 0 50 0 17 0Alcaligenes faeces 0 0 0 0 8 0

ACGA concentrations in rats jaundiced for oneweek this was not a consistent finding and wasnot significantly different from the shamoperated animals at one week and the CDVF1group (Table I).

Bacterial translocationBile duct ligation for both one and three weeksresulted in a significantly increased incidenceof bacterial translocation compared with ratsundergoing sham operation and externalbiliary diversion (Table I). Bacterial trans-location was maximal (11 of 16 animals(68%)) in rats jaundiced for one week andtranslocation was predominantly to themesenteric lymph node complex (MNLC64%, portal blood 18%, liver 18%). Theorganism most often retrieved was Escherichiacoli. In rats jaundiced for a three week periodthe incidence of translocation was similar (sixof 10 animals (60%)) but was more widespreadto other sites - namely, the liver, lungs, andspleen (MNLC 14%, portal blood 14%, liver14%, lung 36%, spleen 22%) with diversity ofthe translocating organism (Table II). Onlyone of six rats undergoing sterile externalbiliary drainage for one week had evidence oftranslocation.

BacteriologyA significant increase in the Gram negativeaerobic population was also noted in the BDL1group and CDVF group compared with thesham l and BDL3 groups (Table I). Both BDLgroups seemed to experience qualitativealteration in the resident caecal microflora witha more diverse pattern of bacterial speciesisolated compared with control groups (TableIII), representing a qualitative disruption of theindigenous microflora.

30 _ No operationo Sham

m20 a BDL

0)

0 5 10 15 20 2

Time from operation (days)Figure 1: Weight change (g (SEM)) in the bile ductligation (BDL) and control rats (no operation and sham)over the period of the experiment.

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.....1 ..

DEL .'1

Figure 2: Low power light micrographs representative of theterminal ileum of the BDL rats (A) and sham operated rats(B). There is considerable blunting of the villi with loss ofvillus height in thejaundiced rat intestine compared withthe control group. (C) Higher power view of the Peyer'spatch at the base of the intestinal crypt in a jaundiced rat. Itshows engorgement ofa Peyer's patch with an increase inthe number of mitotic figures (circles) in the lamina propriaof the terminal ileum.

Apart from the qualitative and quantitativechanges described there were no other differ-ences detected in the caecal microflora.

HistologyHistological analysis of the terminal ileum inthe BDL3 group disclosed non-specificfeatures of small intestinal damage - namely,flattening of the villi with enlargement ofPeyer's patches and an increase in the numberof mitotic figures (Fig 2A, B, and C). Therewas no evidence of mucosal injury in either thesmall or large bowel of the other groups. Thesehistological changes are purely descriptive;however, refined structural and ultrastructuralmorphometric image analysis of the smallintestine is presently underway in our labora-tory.

DiscussionJaundiced patients undergoing invasive diag-nostic and therapeutic procedures are at sig-nificant risk of serious perioperative compli-

cations and death.'1'6 Gram negative sepsisconstitutes the bulk of this morbidity andmortality, but renal dysfunction, coagulopathy,deficient wound healing, and gastrointestinalhaemorrhage are well recognised.17-20The pathophysiology of the complications

seen in extrahepatic biliary obstruction remainsunresolved, but there is increasing evidence tosupport the role of systemic endotoxaemia.Since Wardle and Wright first demonstrated anassociation between renal dysfunction andendotoxaemia in a cohort of jaundiced patientsnumerous authors have concurred with thisfinding in both the clinical and experimental

21-24setting.Endotoxin is a heat stable constituent of the

outer wall of Gram negative bacteria with an0-specific side chain and a highly conservedinner core, which, when present in the systemiccirculation, has the capacity to initiate a farreaching physiological response through therelease of vasoactive inflammatory cytokinesfrom cells of the mononuclear phagocyticsystem.25 27 Although exposure to endotoxin isimportant for the development of the hosts'imune response, when exposed to highsystemic concentrations the effects areexclusively detrimental. There have been twomain hypotheses for the mechanism of sys-temic endotoxaemia in obstructive jaundice. Inthe mammalian system the largest reservoir ofGram negative organisms is the gastrointestinaltract and more specifically the colon. Undernormal physiological circumstances the gutmucosal barrier is impervious to the passage ofbacteria into the sterile environment of theperitoneal cavity, portal circulation, and otherorgan systems such as the liver, lungs, andspleen. The population of Kupffer cellscomprises the bulk of the mononuclearphagocytic system and these are strategicallylocated at the confluence of portal venousdrainage for the sequestration and eliminationof bacteria and portal endotoxins.Any functional or structural disturbance in

either the intestinal mucosal barrier or Kupffercell population will result in the escape ofbacteria and endotoxin to the portal circulationand subsequently to systemic sites, which arenormally sterile. This phenomenon was coined'bacterial translocation' and has been shown tooccur under the influence of various patho-logical insults.28-31 Although there are proteancircumstances in which bacterial translocationhas been shown to occur, several unifyingfactors are implicated in its pathophysiology -namely, physical injury to the intestinalmucosa, bacterial overgrowth, host immunedysfunction, and endotoxaemia.32-34

Physical intestinal injuryIn 1987, Bergesen et al carried out anexperiment in which rats underwent biliarydiversion by way of a choledochocolic fistula.This absence of intraluminal bile salts did notresult in an alteration in villus height, weight,protein, or DNA content in the mucosalscrapings of the terminal ileum although therewas an increase in the excretion of urinary

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Role of the gut in the pathophysiology ofextrahepatic biliary obstruction

indican, an indirect measurement of smallintestinal bacterial overgrowth.35 Morerecently, Deitch et al demonstrated morpho-logical changes in the small intestine in arodent model of biliary obstruction and Dinget al described non-specific abnormalities in themucosa of the small intestine.' 10 This physicalinjury may contribute to breakdown of gastro-intestinal barrier function and consequentlypromote bacterial translocation. Deitch et alhave found this phenomenon in rats to whichendotoxin has been given parenterally3` andattributed these morphological changes to aselective ischaemia-reperfusion injury of theileocaecal segment of the gastrointestinaltract36 effected via the xanthine oxidase path-way. He showed that this effect could beabrogated using the specific xanthine oxidaseinhibitors allopurinol and ibuprofen.37 Otherthan this preliminary data there is noconclusive evidence supporting the role ofphysical injury to the intestinal mucosa as theprimary insult promoting bacterial trans-location in biliary obstruction.

Absence of intraluminal bile and microfloraldisturbancesBurke et al investigated the effects of biliarydiversion on intestinal microflora from thestomach, mid-jejunum and caecum.38 Animalswere pair fed and caged on wire mesh to limitcoprophagy. They were killed after five days ofexternal biliary diversion and although therewas an increase in coliforms and Proteusspecies, this was most pronounced in theproximal small intestine and was found in therats undergoing biliary diversion and pair fedanimals compared with the control group.Their data were interpreted as an effect ofsemistarvation as opposed to the loss ofintraluminal bile salts. Both Deitch et al andDing et al have demonstrated a quantitativedisturbance in the caecal microflora in therodent model of biliary obstruction andSlocum et al intimated that this disturbance inthe Gram negative aerobic population wasattributable to the loss of the constrainingeffects of luminal bile salts.9 10 39 Most of thebacteriological studies on the effects of biliaryobstruction or diversion on the indigenousintestinal microflora have been carried out inrodent models, which may not directly reflectwhat happens in humans. Rats are copro-phagic, which means that the upper smallbowel will not be sterile and over 90% ofsecretory IgA is secreted in the bile; bothcharacteristics are particular to rodents. Theseare important considerations when interpretingresults of bacteriological studies performed inrodent models.

Immune dysfunctionIt is well recognised that extrahepatic biliaryobstruction induces specific and non-specificdepression in host immune status; however, themechanism for this remains unclear.40A3 Thereare many reports of cellular immune dysfunc-tion and Greve et al suggested that depression

ofT cell function was a direct effect of systemicendotoxaemia.44 Gautreaux et al have shownthat T lymphocytes situated in the gastro-intestinal mucosa, Peyer's patches, and mesen-teric lymph node complex have an importantrole in the prevention of bacterial trans-location.45 It has also been shown that T cellmitogen suppression occurs as a secondaryeffect of bacterial translocation.46 There is alarge body of evidence showing impaired mono-nuclear phagocytic system (MPS) function inobstructive jaundice47 48 and although macro-phages are implicated in the transport of viablemicroorganisms from the gut lumen49 to themesenteric lymph node complex, liposomemediated macrophage elimination has beenshown to promote bacterial translocation in thezymosan model. Interestingly, this produced areduction in overall mortality, probablythrough the abrogation of endotoxin mediatedcytokine release from the mononuclear cellpopulation.50

EndotoxaemiaEndotoxaemia has been reported as a sporadic,inconsistent finding in obstructive jaundice;however, with the refinements in the Limuluslysate chromogenic assay5` and the develop-ment of the EndoCab assay for the directmeasurement of antibodies produced to thehighly conserved inner core region ofcirculating endotoxins`3 there is little doubtthat endotoxaemia occurs and has a key role inthe pathophysiology of biliary obstruction. Thecoexistence of these physiological states inbiliary obstruction may well promote bacterialtranslocation.When examining the factors involved in

bacterial translocation it is important toconsider the role of intraluminal constituents,such as bile salts, which have a constrainingrole on the indigenous colonic microflorathrough their emulsifying properties serving todetoxify lumenal endotoxins and maintain thehomeostatic pattern of the indigenous flora.52Secretory IgA is present in large concentrationsin bile, particularly in the rodent, and itsabsence from the gastrointestinal tract mayresult in a disturbance in the local immuno-logical milieu.53 4

The bacterial defences of the bowel reside intwo interesting properties of the colonicbacterial flora. Firstly, the ability to maintaincommunal stability (bacterial antagonism),which involves competition for nutrients,adhesion sites, and the production of anti-microbial factors.55 Secondly, normal intestinalflora can prevent colonisation by exogenousbacteria (colonisation resistance). The obligateanaerobes are thought to play an importantpart in colonisation resistance due to their closeassociation with the intestinal epitheliumconstituting a barrier limiting attachment ofpotential pathogens to the mucosal epi-thelium.56 Colonic bacteria can produce shortchain fatty acids (acetate, proprionate, andbutyrate), all of which provide fuel for thecolonocyte.57 To date most authors haveemployed standard bacteriological techniques

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to quantify and qualify bacterial translocation,although it would seem from recent reportsthat it may not be sufficient to characterisemicroorganisms solely by their species.58 It hasbeen shown using biochemical fingerprintingthat certain bacterial phenotypes have apropensity for translocation and it may bemore important to assess the qualitativeconstitution of specific bacterial species in thefaecal flora as opposed to isolated alterations inquantitative counts.59The aim of this study was to evaluate the

effects of increasing duration of biliary obstruc-tion on bacterial translocation in relation totemporal alterations in the indigenous micro-ecology over a three week period.

Bile saltsBile salts have been shown to inhibit thegrowth of intestinal bacteria in both theexperimental and clinical setting.` ` Themechanism is unclear. However, this has beenattributed to their detergent action in whichthe bacterial cell membrane integrity iscompromised and the endotoxin molecule isbroken down into inert subunits or formedunabsorbable micellar aggregates.63 In clinicalstudies oral treatment with bile acids has beenshown to reduce systemic endotoxaemia andthe incidence of renal failure in jaundicedpatients.64 65 Ding et al also showed a reductionin bacterial translocation in jaundiced animalsto which bile salts were administeredenterally.66

Study commentaryBacterial translocation was demonstrable inanimals jaundiced for both one and threeweeks. The shorter period of biliaryobstruction resulted in significant overgrowthofGram negative aerobic bacteria and a greaterincidence of translocation, although this waspredominantly to the mesenteric lymph nodecomplex. With increasing duration of bile ductligation, animals had a lower incidence ofbacterial translocation and no significantquantitative disturbance in the microecologicalfloral pattern. Despite this, a broader range ofvegetative species were isolated from thecaecum with wider dissemination to the portalblood, liver, lungs, and spleen. Consistent withthe increased systemic exposure, high plasmaconcentrations of anticore glycolipid antibodyand endotoxin were demonstrable. In theanimals jaundiced for a three week period thesite where most translocation was found wasthe lungs and these data support theobservation of Katz et al, who recognised thatin the absence of normal Kupffer cell functionthe pulmonary system compensates withgreater bacterial trapping.60 Pulmonaryalveolar macrophages (PAMs) have lessbactericidal potential than Kupffer cells, whichmay permit bacteria to survive in a state ofintracellular symbiosis resulting in activation ofthe PAM, perpetuating the inflammatoryresponse in the lungs. These data concur withthe findings of Ding et al and raise the

possibility that there may be two distinctmechanisms responsible for the bacterialtranslocation seen at the different time pointsafter ligation of the bile duct.

After one week of bile duct ligation absenceof intraluminal bile salts results in the over-growth of Gram negative aerobes, which is themost likely mechanism for translocation. Thelimitation of translocation to the mesentericlymph node complex and the low plasma con-centrations of endotoxin and anticore glyco-lipid antibodies in the BDL1 group may reflectnormal mononuclear phagocytic function atthis stage of biliary obstruction. In the CDVFgroup, in which bile flow was externallydiverted from the gastrointestinal tract in theabsence of biliary obstruction there was asimilar overgrowth in the Gram negativeaerobic population. No bacterial translocationwas demonstrable in this group despite themicroecological disturbances, althoughSlocum et al showed not only disturbances inlumenal floral counts but also bacterialtranslocation in rats undergoing CDVF,reinforcing the argument that bacterialtranslocation seen in the early stages of biliaryobstruction probably is directly related to adisturbance in the indigenous microfloraoccurring as a consequence of the absence ofintraluminal bile salts.With the increasing duration of biliary

obstruction the intraluminal microecologicaldisturbances are no longer demonstrable. It isplausible that with increased duration of biliaryobstruction the systemic concentrations of bileacids increase proportionately and may diffuseback to the intestinal tract exerting their homeo-static effects on the intestinal flora. Despite thisnormalisation in the microflora the efficiencyof the MPS, specifically the Kupffer cellpopulation, to sequester and clear endo-toxaemia is impaired. Depression in thephagocytic potential of Kupffer cells may resultfrom physical effects of biliary obstruction andraised intraductal pressure, the toxic effects ofhigh bile salt concentrations, portal endo-toxins, and inflammatory mediators locally inthe liver. This is reflected in increased concen-trations of endotoxin and anticore glycolipidantibody systemically and the broader patternof bacterial translocation to other organsystems. Depression of host immune functionwith the concomitant effects of systemic endo-toxaemia on gastrointestinal mucosal integritymay be responsible for bacterial translocationseen in the later stages of biliary obstruction.

In conclusion, we postulate that absence ofintraluminal bile salts results in bacterialovergrowth and bacterial translocation in theearly stages of biliary obstruction. Withprogression of biliary obstruction there is lossof the constraining effects ofMLNC on entericbacteria resulting in systemic dissemination.This may well be related to deficient mono-nuclear phagocytic function. With the 'spill-over' of portal endotoxin systemically, there isa circuitous effect on gastrointestinal mucosalintegrity resulting in further bacterialtranslocation and perpetuation of the systemicseptic inflammatory response.

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